Various inflammatory diseases are characterized by leakage induced edema. On a cellular level, this edema is associated with the junctions between epithelial or endothelial cells being compromised. One such mechanism related to this type of inflammation is ischemic tissue compensating for a lack of oxygen & nutrients by excessively up-regulating cytokines involved in permeability. The over-expression of vascular endothelial growth factor (VEGF) causes an increase in vessel permeability due to depletion of cell-to-cell adhesion molecules such as VE-cadherin and claudin-5. Crohn's disease and inflammatory bowel disease have been shown to progress through a failed tight junction mechanism. (J D Schulzke et al. Ann N Y Acad Sci. 2009 May; 1165:294-300. doi: 10.1111).
Endothelial tight junctions are found in a variety of tissues including blood vessels and the brain, the endothelial lining of the vessel wall forms a controlled permeable barrier, which is located at the interface between the vascular and the perivascular compartments. Although the endothelium acts as an efficient barrier that strictly separates the two compartments, it may also act as a permeable filter which allows selective exchange of solutes and water between the luminal and abluminal sides of the barrier. A disruption of the equilibrium function through tight junction failure leads to a variety of inflammatory conditions of the vasculature, lymphatic system, and brain. While such conditions are routinely treated with steroidal or non-steroidal anti-inflammatories, such existing therapies have limitations in terms of efficacy, as well as side-effects.
Additionally, many cancers progress only through angiogenesis that is promulgated by vascular endothelial growth factor (VEGF) and metastasis of tumors necessarily requires the loosening of junctions between cancerous cells to allow cells to become circulatory. While not causative of cancer, this mechanism seems to be important in proliferation.
The epithelial and endothelial layers are sites of exchange as well as barriers, for the transit of ions and molecules between tissues and the circulatory system of the organism. Complexes between adjacent epithelial and endothelial cells include Tight Junctions and Adherens junctions. Vertebrate epithelial and endothelial cells exhibit Tight Junctions that lie apical to Adherens Junctions, Tight Junctions have an organizing role in epithelial and endothelial polarization and establish an apico-lateral barrier to the diffusion of solutes through the intracellular space (gate function). Tight junctions also restrict the movement of lipids and membrane proteins between the apical and the basolateral membrane (fence function). Tight Junctions are highly ordered membrane contact sites, comprising a network of intra-membrane fibrils. Tight Junctions include transmembrane proteins, including occludin, claudin-5, and junctional adhesion molecules (JAMs), and a number of cytoplasmic peripheral proteins. These are shown schematically in prior art FIG. 1. While the transmembrane proteins mediate cell-cell adhesion, the cytosolic tight junction plaque contains various types of proteins (e.g. PDZ proteins, such as the ZO (Zona Occludens) family) that link tight junction transmembrane proteins to the underlying cytoskeleton. These adapters also recruit regulatory proteins, such as protein kinases, phosphatases, small GTPases and transcription factors, to the tight junctions. As a result, structural (Actin and Spectrin) and regulatory (Actin-binding proteins, GTPases and kinases) proteins are juxtaposed with transmembrane proteins. This protein scaffolding facilitates the assembly of highly ordered structures, such as junctional complexes or signaling patches that regulate epithelial cell polarity, proliferation and differentiation.
Tight Junctions are located at the uppermost portion of the lateral plasma membrane, where the integral membrane proteins like claudins appear to be involved in the homophilic and/or heterophilic interactions implicated in firm adhesions. Claudins have four hydrophobic transmembrane domains and two extracellular loops. The extracellular loops, whose sequences are distinct in different claudins, contribute to the formation not only of tight junction strands but also of ion-selective channels. Claudin-5 is important in endothelial and epithelial cell junctions. In general, tight junction strands are linear co-polymers of occludin, claudin-5, and JAMs that attract cytoplasmic proteins containing PDZ domains (OZ) have high affinity for the C-terminal sequences of these proteins.
Tight Junctions and Adherens Junctions are functionally and structurally linked, endothelial VE-cadherin associated with Adherens Junctions upregulates the gene encoding the Tight Junction adhesive protein claudin-5 and a similar structure is found in epithelial cells with E-cadherin in place of VE-cadherin. This effect requires the release of the inhibitory activity of forkhead box factor FoxO1 to suppress proteasome activity. Vascular endothelial (VE)-cadherin acts by inducing the phosphorylation of FoxO1 through Akt activation and by limiting the translocation of beta-catenin to the nucleus. (Taddei et al. Nat Cell Biol. 2008 August; 10(8):923-34. doi: 10.1038/ncb1752. Epub 2008 Jul. 6). Polycystin-1 (PDK-1) is a membrane protein localized to Adherens Junctions in a complex containing beta-catenins, that is mediated by P13K.
VEGF induces vascular permeability through induction of the rapid endocytosis of a key endothelial and epithelial cell adhesion molecule, Cadherin, thereby disrupting the barrier function. This process is initiated by the activation of the small GTPase, Rac by VEGFR through the Src-dependent phosphorylation of Vav2 (not shown), a guanine nucleotide-exchange factor. Rac activation, in turn, promotes the p21-activated kinase (PAK)-mediated phosphorylation of a highly conserved motif within the intracellular tail of Cadherin. This results in the disassembly of intercellular junctions. (Gavard et al., Nat Cell Biol. 2006 November; 8(11):1223-34. Epub 2006 Oct. 22).
In a normally functioning exemplary epithelial cell shown in the left panel of FIG. 1 with an intact Tight Junction, VEGF is not bound to its corresponding receptor VEGFR, and claudin-5 is expressed normally in the nucleus from the encoding claudin-5 gene and processed by the endoplasmic reticulum. The occludin, claudin-5, and JAM together form a functioning Tight Junction, and Cadherin forms and organized Adherens Junction.
In contrast, with VEGF binding to VEGFR, as shown in the right panel of FIG. 1, the Src/Rac/Pak complex acts on beta-catenins to destabilize the Adherens Junction. The resultant cascade is believed to disrupt claudin-5 expression and assembly resulting in a loss of Tight Junction structure.
Norrin is a ligand for the Frizzled receptor subtype 4 (Fz4). Norrin hinds Fz4 with nanomolar affinity (Xu, et al, Cell, 2004; 116:883-895; Clevers, Curr Biol, 2004; 14:R436-437; Nichrs, Dev Cell, 2004; 6:453-454). Norrin interaction with Fz4 is dependent on the cell surface receptor LRP5. (Xu, 2004). Frizzled receptors are coupled to the β-catenin canonical signaling pathway. The inactivation of glycogen synthase kinase (GSK) 3β and Axin through frizzled receptor binding stabilizes β-catenin, which subsequently accumulates in the cell nucleus and activates the transduction of target genes that are crucial in the G1-S-phase transition, such as cyclin D1 or c-Myc. (Willert et al., Curr Opin Genet Dev, 1998; 8:95-102). Suppression of norrin activity has been shown to preclude angiogenesis associated with ocular disease (US 2010/0129375).
Thus, there exists a need for a method to treat edema associated with endothelial and epithelial membrane leakage. There further exists a need for a method to treat clinical disorders associated with endothelial and epithelial cell membrane failure edema. There also exists a need for a method to produce cadherin and claudin-5. The present invention is directed to these, as well as other, important needs in the art.